Antihuman thymidylate synthase monoclonal antibodies and hybridomas producing the same

ABSTRACT

This invention relates to an anti-human thymidylate synthase monoclonal antibody capable of recognizing an epitope which exists in a region of 187th to 313th amino acids from an N-terminus in human thymidy late synthase, an anti-human thymidylate synthase monoclonal antibody capable of recognizing an epitope which exists in a region of from an N-terminus to a 61st amino acid in human thymidylate synthase, and also hybridomas capable of producing these monoclonal anti-bodies. 
     These monoclonal antibodies are useful for the immunological measurement of human thymidylate synthase.

TECHNICAL FIELD

This invention relates to a novel monoclonal antibody to humanthymidylate synthase and also to a hybridoma capable of producing thesame.

BACKGROUND ART

Thymidylate synthase (EC2.1.1.45, hereinafter called “TS”) is an enzymethat catalyzes a reaction in which thymidylic acid is formed fromdeoxyuridylic acid, plays a role to supply thymine which is a basespecific to DNAs, and is one of principal rate-limiting enzymes for aDNA precursor supply pathway. Accordingly, its activity is known tobecome higher in thymi or tumor tissues where cell growth is active.

On the other hand, fluoropyrimidine antitumor drugs such as5-fluorourasil and 5-fluorodeoxyuridine act against TS as a targetenzyme, and for example, 5-fluorodeoxyuridine changes intofluorodeoxyuridylic acid in vivo and inhibits TS. In particular,fluoropyrimidine antitumor drugs are known to show high therapeuticeffect and significant life prolongation for patients with a low levelof TS expression in tumor cells but to exhibit low therapeutic effectfor patients with TS in a high level [“Gan to Kagaku Ryoho (Cancers andChemotherapy)”, 24(6), 705-721 (1997)]. The measurement of TS istherefore important, for example, an advance measurement of theexpression of TS in an excised tumor upon treatment for a cancer patientgives indications for the determination of a treatment method and forthe selection of an antitumor drug.

An object of the present invention is therefore to provide an anti-humanTS monoclonal antibody useful for the immunological measurement of humanTS and also a hybridoma capable of producing the same.

DISCLOSURE OF THE INVENTION

With the foregoing circumstances in view, the present inventors haveproceeded with various investigations. As a result, novel anti-human TSmonoclonal antibodies have been obtained and moreover, these monoclonalantibodies have been confirmed to be useful for the immunologicalmeasurement of human TS, leading to the completion of the presentinvention.

The present invention therefore provides an anti-human TS monoclonalantibody capable of recognizing an epitope which exists in a region of187th to 313th amino acids from an N-terminus in human TS, an anti-humanTS monoclonal antibody capable of recognizing an epitope which exists ina region of from an N-terminus to a 61st amino acid in human TS, andalso hybridomas capable of producing these monoclonal antibodies.

Incidentally, the recognition regions by the human TS monoclonalantibodies according to the present invention have been determined inaccordance with the amino acid sequence of human TS described, forexample, in Nucleic Acids Research, 13(6), 2035-2043 (1985) or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 electrophoretogrammatically shows recognition regions in TS bymonoclonal antibodies according to the present invention.

FIG. 2 electrophoretogrammatically illustrates detection results ofhuman TS by Western blotting in which the monoclonal antibodiesaccording to the present invention were used.

FIG. 3 diagrammatically depicts measurement results of human TS by ELISAin which the monoclonal antibodies according to the present inventionwere used.

BEST MODES FOR CARRYING OUT THE INVENTION

Illustrative of the anti-human TS monoclonal antibody according to thepresent invention, which can recognize the epitope existing in theregion of 187th to 313th amino acids from the N-terminus in human TS,are those produced by mouse hybridomas RTSMA1 (FERM BP-6404) and RTSMA2(FERM BP-6402) obtained using, as an immunogen, a recombinant human TS(hereinafter called “rhTS”) to be described subsequently herein. Thesehybridomas have been deposited under the Budapest Treaty in NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry(address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-0046,JAPAN) (date of original deposit: Jun. 30, 1998).

The above rhTS can be produced as will be described hereinafter. Namely,a plasmid from which a glutathion S-transferase (GST)-TS fused proteincan be derived by isopropyl-1-thio-β-D-galactoside (hereinafter called“IPTG”) is prepared by inserting restriction enzyme recognition sitesMunI to HindIII of cDNA of human TS into a plasmid designed to expressthe GST-TS fused protein. An Escherichia coli strain which has beentransformed with the plasmid is mass-cultured in the presence of IPTG,and the crude GST-TS is loaded onto a glutathion-agarose column. Thethus-adsorbed GST-TS fused protein is eluted with a suitable buffer, andis then incubated in the presence of thrombin and calcium chloride tocleave human TS and GST from each other. The resulting mixture is loadedfurther onto a GST-agarose column, whereby rhTS of high purity can beobtained.

On the other hand, examples of the anti-human TS monoclonal antibody ofthe present invention, which can recognize the epitope existing in theregion of from the N-terminus to the 61st amino acid in human TS, caninclude-those produced by mouse hybridomas NTSMA1 (FERM BP-6401) andNTSMA2 (FERM BP-6403) obtained using, as an immunogen, a native human TS(hereinafter called “nhTS”) which is available from the tissues byextraction and purification. These hybridomas have been deposited underthe Budapest Treaty in National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry (address: 1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken 305-0046, JAPAN) (date of original deposit:Jun. 30, 1998).

The above nhTS can be produced based on the method proposed by Rode etal. [Rode et al., Biochemical Pharmacology 29, 723 (1980)] as will bedescribed hereinafter. Namely, nhTS of high purity can be obtained byloading a homogenate of incubated cells of a human lung cancer strainLu-99 or of a tumor, which has been excised from a nude mouse or nuderat with sub-cutaneously transplanted Lu-99, onto a column with ethyl10-formyl-5,8-dideazafolate immobilized therein as a ligand and theneluting nhTS with a buffer containing dUMP.

Each of the hybridomas according to the present invention can beobtained by immunizing a mammal, such as a mouse or a rat, or a birdwith the rhTS or nhTS, subjecting its spleen cells and myeloma cells ofa mammal, such as a mouse or a rat, to cell fusion in accordance withthe method originally outlined by Kohler and Milstein [see Nature, 256,495 (1975)], and then culturing the fused cells in a selection medium.The hybridomas RTSMA1 and RTSMA2 are obtained when rhTS is used as animmunogen, while the hybridomas NTSMA1 and NTSMA2 are obtained when nhTSis used as an immunogen.

No particular limitation is imposed on an immunization method.Immunization can be achieved, for example, by dissolving rhTS or nhTS inphosphate buffer, physiological saline or the like, mixing an adjuvantas needed, and then administering the resulting solution several timesat intervals of 1 to 3 weeks under the skin or into the spleen orperitoneal cavity or a vein of the animal. Illustrative of myeloma cellsusable for cell fusion are mouse P3-NS-1/1Ag4.1, P3-X63-Ag8.653 andSP2/0Ag14 and rat YB2/0. Upon cell fusion, polyethylene glycol, Sendaivirus or the like can be used as a fusion promoter, or electric pulsesmay be used.

The anti-human TS monoclonal antibodies according to the presentinvention can each be produced by culturing the corresponding one of theabove-described hybridomas in a suitable culture medium or within theperitoneal cavity of a mammal (for example, mouse). An illustrativemedium suitably usable for the incubation of the hybridoma is a mediawhich contains bovine fetal serum, L-glutamine, L-pyruvic acid, andantibiotics (penicillin G and streptomycin) in Dulbeccos modifiedEagle's minimum essential medium. The incubation of the hybridoma isconducted, for example, in a 5% CO₂ concentration at 37° C. for 3 dayswhen it is performed in a culture medium or, for example, for 14 dayswhen it is performed within the peritoneal cavity of a mouse. From theculture medium or mammalian ascites obtained as described above, thecorresponding anti-human TS monoclonal antibody RTSMA1, RTSMA2, NTSMA1or NTSMA2 according to the present invention can be separated andpurified by a method commonly employed for the isolation and/orpurification of proteins. Examples of such a method can includesalting-out with ammonium sulfate, ion-exchange column chromatographymaking use of an ion-exchange cellulose, molecular sieve columnchromatography making use of a molecular sieve gel, affinity columnchromatography making use of polysaccharide complexed with protein A,dialysis, and lyophilization.

The anti-human TS monoclonal antibodies of the present inventionobtained as described above are useful for the immunological measurementof human TS, and can be applied, for example, to the sandwich method,competitive radioimmunoassay, enzyme immunoassay andimmunochromatography. When plural ones of the monoclonal antibodiesaccording to the present invention are used in combination in thesemeasuring methods, combinations of antibodies the epitopes of which areapart are preferred for obtaining high absorbances. It is thereforepreferred to combine RTSMA1 or RTSMA2 with NTSMA1 or NTSMA2.

Further, a combination of an anti-human TS polyclonal antibodyimmobilized on an insoluble carrier makes it possible to easily measurehuman TS with high accuracy. Such an anti-human TS polyclonal antibodycan be obtained in a manner known per se in the art by administeringnhTS or rhTS to a suitable mammal such as mouse, rat, rabbit or sheep.

EXAMPLES

The following examples are presented to illustrate the present inventionin further detail, but it is to be understood that the present inventionis not limited thereto.

Referential Example 1

A. Preparation of rhTS

An Escherichia coli strain NM522, in which a plasmid prepared withrestriction endonuclease recognition sites MunI to HindIII of human TScDNA inserted therein to express a fused protein of glutathioneS-transferase (GST) and human TS was incubated overnight at 37° C. undershaking in LB medium (200 mL) (product of Wako Pure Chemical Industries,Ltd.) in the presence of ampicillin (50 ug/mL). The culture medium waspoured in 100 mL aliquots into two Erlenmeyer flasks which containedampicillin-containing LB medium (1 liter/flask). They were incubated at25° C. for 3 hours under shaking, to which 0.6 mL aliquots ofisopropyl-1-thio-β-D-galactoside (IPTG, 40 mg/mL) were addedrespectively, followed by further incubation at 25° C. for 20 hours.cells were collected by centrifugation and were then suspended in adisrupting buffer (100 mL; 50 mM Tris, pH 7.5, 25% sucrose). “10%Nonidet P-40” (5 mL; sur- factant, product of NACALAI TESQUE INC.) and 1M magnesium chloride (0.5 mL) were added. The cells were disrupted by asonicator, followed by centrifugation at 10,000 rpm for 15 minutes. Thesupernatant was caused to pass (20 mL/hr) through a column packed withglutathione (GSH)-agarose (14 mL; product of Sigma Chemical Co.). Afterthe column was washed with a wash (100 mL; 20 mM Tris, pH 7.5, 2 mMmagnesium chloride, 1 mM DTT), the column was eluted with an eluent (50mL; 50 mM Tris, pH 9.6, 5 mM GSH) such that the eluate was received in3-mL aliquots in tubes. By confirming protein fractions in accordancewith the Bradford's method, peak fractions (9 mL; protein concentration:7 mg/mL) were obtained. They were immediately dialyzed against a wash (1liter) to lower their pH back to 7.5, and thrombin (600 units) wasadded. The mixture so prepared was treated at 37° C. for 2 hours in thepresence of 1 mM calcium chloride, whereby the GST-TS fused protein wascleaved at bound sites. The resulting mixture of GST and TS was againcaused to pass through a GSH-agarose column (20 mL/hour), the column waseluted with a wash, and protein fractions were confirmed by theBradford's method, whereby an rhTS solution (9 mL) was obtained. 0.2,0.4, 0.6, 0.8 and 1.0 mg/mL BSA solutions (100 μL) were added to 5-mLaliquots of the Bradford's solution, respectively, and their absorbancesat 595 nm were measured to prepare a standard curve. An rhTS solution(100 μL), which had been diluted fivefold in distilled water, was addedto the Bradford's solution (5 mL) and the absorbance at 595 nm wasmeasured. As a result, the protein concentration of the rhTS solutionwas found to be 3.5 mg/mL.

B. Preparation of nhTS

Purification of nhTS was conducted based on the method proposed by Rodeet al. [Rode et al., Biochemical Pharmacology, 29, 723 (1980)]. A humanlung cancer strain Lu-99, which had been subcutaneously transplanted todorsal regions of 50 male BALB/c-nu/nu mice, was removed to obtaintumors (50 g). Those tumors were added with 10 mM phosphate buffer (100mL; pH 7.5, 100 mM potassium chloride, 10 mM 2-mercaptoethanol) and werethen homogenized. The homogenate was centrifuged at 4° C. and 10,000 rpmfor 1 hour, and from the supernatant, a precipitate was obtained withammonium sulfate at 30-70% saturation. The precipitate was dissolved in10 mM phosphate buffer (pH 7.5, 0.1% Triton X-100, 10 mM2-mercaptoethanol, 20 μM dUMP). The resulting solution was loaded onto acolumn while using ethyl 10-formy1-5,8-dideazafolate as a ligand. Afterthe column was washed with 200 mM phosphate buffer (pH 7.5, 0.1% TritonX-100, 10 mM 2-mercaptoethanol, 20 μM dUMP), the column was eluted with200 mM phosphate buffer (pH 7.5, 0.1% Triton X-100, 10 mM2-mercaptoethanol, 20 μM dUMP) and an nhTS solution (4 mL) was obtained.0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL BSA solutions (100 μL) were added to5-mL aliquots of the Bradford's solution, respectively, and theirabsorbances at 595 nm were measured to prepare a standard curve. An nhTSsolution (100 μL) was added to the Bradford's solution (5 mL) and theabsorbance at 595 nm was measured. As a result, the proteinconcentration of the nhTS solution was found to be 0.3 mg/mL.

Example 1

Preparation of Monoclonal Antibodies RTSMA1 and RTSMA2

To a female BALB/c mouse (8 weeks old), the rhTS obtained in ReferentialExample 1A was intraperitoneally injected at a dose of 20 μg/mouse. TheTS protein was used in a form emulsified beforehand in Freund's completeadjuvant. To the mouse, the rhTS in a form emulsified beforehand inFreund's incomplete adjuvant was additionally and intraperitoneallyinjected at a dose of 20 μg/mouse four times successively at intervalsof 14 days. Three days before fusion, the rhTS (100 μg) inphosphate-buffered physiological saline (0.5 mL) was injected through acaudal vein. Spleen cells (1×10⁸) from the immunized mouse, P3-X63-Ag8.653 myeloma cells (2×10⁷) and as a fusing reagent, “50% (V/V)polyethylene glycol 4000” (product of Merck & Co., Inc.) were used tofuse those cells together in accordance with the fusing method proposedby Galfre and Milstein [Galfre et al., Nature 266, 550 (1977)].

After the fusion, cells were suspended in HAT medium (RPMI1640 mediumcontaining 1×10³¹ ⁴ M hypoxanthine, 4×10⁻⁷ M aminopterin and 1.6×10⁻⁵ Mthymidine), which contained 10% bovine fetal serum, to give a cellconcentration of 110 ⁶ cells/mL. The resultant suspension was dispensedin 200 μL aliquot per well onto a 96-well microplate.

Fused cells were cultured in a CO₂ incubator (5% CO₂, 37°C.), duringwhich replacements of the medium were conducted using HAT mediumcontaining 10% bovine fetal serum so that the fused cells were allowedto proliferate. A hybridoma formed of the spleen cells and the myelomacells were screened, and was then conditioned in HT medium (RPMI1640medium containing 1×10³¹ ⁴ M hypoxanthine and 1.6×10³¹ ⁵ M thymidine)which contained 10% bovine fetal serum.

The antibody in the incubation supernatant of the hybridoma was detectedin accordance with ELISA by using an rhTS-sensitized microplate. Withrespect to each well which was found to be positive, cloning wasrepeated twice in accordance with the limiting dilution analysis byusing HT medium which containing 10% bovine fetal serum and 5% Bleiclone(product of Dainippon Pharmaceutical Co., Ltd.). Two kinds of cloneswhich had reactivity to the rhTS were hence chosen, and were named“RTSMA1” (FERM BP-6404) and “RTSMA2” (FERM BP-6402).

Pristane (0.5 mL; product of Dainippon Pharmaceutical Co., Ltd.) wasintraperitoneally injected to a nude mouse. Seven days later, pristane(0.5 mL) was intraperitoneally administered further, and the hybridomasRTSMA1 and RTSMA2 (1×10⁷ cells) were transplanted to the peritonealcavity and were allowed to proliferate. After 2 to 3 weeks, ascites wasobtained. The ascites was caused to pass through a “Protein G Sepharose4FF ” column (product of Pharmacia AB). After the column was washed witha wash (20 mM sodium phosphate, pH 7.0), the monoclonal antibodiesRTSMA1 and RTSMA2 produced by the respective clones were eluted with theeluent (0.1 M glycine, pH 2.7) and were immediately dialyzed against awash.

Example 2

Preparation of Monoclonal Antibodies NTSMA1 and NTSMA2

In a similar manner as in Example 1 except that as an immunogen, thenhTS obtained in Referential Example B1 was used in place of the rhTS,two kinds of clones having reactivity to the nhTS were chosen, and werenamed “NTSMA1” (FERM BP-6401) and “NTSMA2” (FERM BP-6403). From thesehybridomas, monoclonal antibodies NTSMA1 and NTSMA2 were then obtainedin a similar manner as in Example 1.

Test 1 Identification of Recognition Sites by the Monoclonal AntibodiesAccording to the Present Invention

Separately transformed in the Escherichia coli strain NM522 wereplasmids which had been prepared to express GST-human TS fused proteinsby inserting the restriction endonuclease recognition sites MunI to SphI(which encode the amino acids of human TS from the N-terminus to the61st amino acid), the restriction endonuclease recognition sites MunI toBglII (which encode the amino acids of TS from the N-terminus to the186th amino acid) and the restriction endonuclease recognition sitesMunI to HindIII (which encode the amino acids of TS from the N-terminusto the 313th amino acid), respectively. The respective Escherichia coliclones were incubated overnight at 37° C. under shaking in LB medium (5mL) (product of Wako Pure Chemical Industries, Ltd.) in the presence ofampicillin (50 μg/mL) and 1 mM IPTG.

The culture medium was transferred in 100 μL aliquots into 1.5 mLcentrifuge tubes. Subsequent to centrifugation at 12,000 r.p.m. for 5minutes, the supernatants were discarded. An electrophoresis samplepreparation solution (100 μL) (4% SDS, 10% β-mercaptoethanol, 20%glycerol, 125 mM Tris-, pH 6.8) was added the resulting pellets tosuspend them. The suspension was heated for 15 minutes on boiling water,and a 10-μL aliquot was then used for electrophoresis. After the samplewas allowed to migrate using 12.5% polyacrylamide gel, the sample somigrated was electrically transferred onto a PVDF filter. The PVDFfilter was then immersed in “BLOCK ACE” (blocking agent, product ofDainippon Pharmaceutical Co., Ltd.) to conduct blocking.

Using as primary antibodies the respective monoclonal antibodiesprepared to 5 μg/mL with 20 mM PBS (pH 7.0), reactions were conductedfor 1 hour. After the filter was washed with 20 mM Tris pH 7.0 (wash)which contained 500 mM sodium chloride and 0.05% Tween 20, reactionswere conducted for 1 hour by using a biotinylated goat anti-mouse IgGantibody (product of VECTASTAIN) as a secondary antibody. The filter wasthen washed with a wash, and “ABC Reagent” (alkaline-phosphatase-labeledavidin-biotin complex solution, product of VECTASTAIN) was reacted.After washing with a wash, enzyme reactions were conducted by using an“Alkaline Phosphatase Substrate KIT II” (product of VECTASTAIN) todetect TS.

As a result, as is shown in FIG. 1, the monoclonal antibodies RTSMA1 andRTSMA2 recognized the TS protein over the entire length thereof, but didnot recognize the reconstructed TS protein ranging from the N-terminusto the 186th amino acid. The antigen recognition sites (epitopes) ofboth of the antibodies were therefore found to exist in the region offrom the 187th to 313th amino acids. On the other hand, the monoclonalantibodies NTSMA1 and NTSMA2 recognized all of the region ranging fromthe N-terminus to the 61st amino acid, the region ranging from theN-terminus to the 186th amino acid, and the reconstructed TS proteinover the entire length thereof. The epitopes of these antibodies werehence found to exist in the region of from the 1st to 61st amino acids.

Test 2 Confirmation of Specificity of Monoclonal Antibodies of thePresent Invention by Western blotting

A cell homogenate of human leukemia derived cell CCRF-CEM (proteinconcentration: 10 mg/mL) was mixed with an equiamount of anelectrophoresis sample preparation solution (4% SFS, 10%β-mercaptoethanol, 20% glycerol, 125 mM Tris, pH 6.8). The mixture washeated for 2 minutes in boiling water, and a 10-μL aliquot was then usedfor electrophoresis. After the sample was allowed to migrate using 12.5%polyacrylamide gel, the sample so migrated was electrically transferredonto a PVDF filter. The PVDF filter was then immersed in “BLOCK ACE”(blocking agent, product of Dainippon Pharmaceutical Co., Ltd.) toconduct blocking.

Using as primary antibodies the respective monoclonal antibodiesprepared to 5 μg/mL with 20 mM PBS (pH 7.0), reactions were conductedfor 1 hour. After the filter was washed with 20 mM Tris pH 7.0 (wash)which contained 500 mM sodium chloride and 0.05% Tween 20, reactionswere conducted for 1 hour by using a biotinylated goat anti-mouse IgGantibody (product of VECTASTAIN) as a secondary antibody. The filter wasthen washed with a wash, and “ABC Reagent” (product of VECTASTAIN) wasreacted. After washing with a wash, enzyme reactions were conducted byusing an “Alkaline Phosphatase Substrate KIT II” (product of VECTASTAIN)to detect TS.

As a result, as is shown in FIG. 2, the monoclonal antibodies accordingto the present invention were all confirmed to specifically recognizeonly TS.

Test 3 Reactivity of the Monoclonal Antibody of the Present Invention torhTS An rhTS solution, which had been adjusted to 10 μg/mL with 50 mMcarbonate buffer (pH 9.5), was poured in 0.1 mL aliquots into therespective wells of a 96-well plate (“Immunoplate II Maxisope”, productof Nunc). While keeping the plate sealed, coating was conducted for 1hour to immobilize the antibody. The 96-well plate was washed twice witha wash (physiological saline containing 0.05% Tween 20), “BLOCK ACE”(blocking solution, product of Dainippon Pharmaceutical Co., Ltd.) waspoured in 200 μL aliquots into the respective wells, and blocking wasthen performed for 1 hour at sites where the antibody wasnon-specifically adsorbed.

The blocking solution was then discarded, and as primary antibodies, therespective monoclonal anti-bodies (0.1 mL) which had been adjusted to0.00244 to 5 μg/mL by diluting 5 μg/mL, as an initial concentration ofdilution, twofold in 20 mM PBS (pH 7.0) were added and then reacted for1 hour. The resulting mixture was washed twice with a wash, and thebiotinylated goat anti-mouse IgG antibody (100 μL) (product ofVECTASTAIN) was added, followed by reactions for 1 hour as in the caseof the primary antibodies. After completion of the reactions, thereaction mixture was washed twice with a wash. The ABC reagent (100 μL)(product of VECTASTAIN) was added, and subsequent to completion ofreactions for 1 hour, the reaction mixture was washed four times with awash. A diethanolamine buffer (0.1 mL) (product of Kirkegaard and PerryLaboratories which contained 1 mg/mL paranitrophenylphosphate was added,followed by enzyme reactions for 10 minutes. 0.1 mL aliquots of 0.1 Msodium hydroxide were added to terminate the reactions, and measurementswere conducted with the measuring absorbance of an ELISA plate readerset at 405 nm.

As a result, as is shown in FIG. 3, the monoclonal antibodies RTSMA1,RTSMA2, NTSMAL and NTSMA2 gave absorbances of 0.3 or higher (background:0.08) when used at concentrations of 0.08, 0.08, 0.02 and 0.16 μg/mL,respectively, and were able to achieve detection of rhTS at theseconcentrations.

Test 4 Comparison of Antibody Combinations by the Sandwich Method

The monoclonal antibodies RTSMA1 and RTSMA2 were adjusted to 2 μg/mLwith 50 mM carbonate buffer (pH 9.5) and then dispensed in 0.1 mLaliquots onto a 96-well ELISA plate. The plate was sealed and was thensubjected to coating for 2 hours in an incubator controlled at 37° C.,whereby an antibody-immobilized carrier was obtained. After the carrierwas washed twice with a wash (physiological saline containing 0.05%Tween 20), rhTS which had been adjusted to 10 μg/mL with 20 mM PBS(diluting solution) containing 0.05% Tween 20 was dispensed in 0.1 mLaliquots onto the antibody-immobilized plate and was then staticallyreacted at 37° C. for 1 hour.

After the wells were washed twice with a wash, the enzyme-labeledproducts of the monoclonal antibodies bodies RTSMA1, RTSMA2, NTSMAl andNTSM2 which had been adjusted to 1 μg/mL with the diluting solution weredispensed in 0.1 mL aliquots, and were statically reacted at 37° C. for1 hour. After the wells were washed four times with a wash, 0.1 mLaliquots of 0.1 M acetate buffer (pH 5.5; color-developing solution)which contained 3 mg/mL orthophenylenediamine and 0.75 mM hydrogenperoxide were added, and enzyme reactions were allowed to proceed atroom temperature for 30 minutes in a dark place. Finally, 0.1 mLaliquots of 0.1 M sulfuric acid were added to terminate the reactions,and measurements were conducted with the measuring absorbance of anELISA plate reader set at 490 nm. The results are presented in Table 1.

TABLE 1 Differences in Absorbance (490 nm) by Combinations of AntibodiesEnzyme-labeled antibody Immobilized antibody RTSMA1 RTSMA2 NTSMA1 NTSMA2RTSMA1 0.4 0.4 7.2 4.0 RTSMA2 0.8 0.4 8.0 4.2

As is apparent from Table 1, absorbance is low when monoclonalantibodies the epitopes of which are close to each other are used as animmobilized antibody and an enzyme-labeled antibody, but a highabsorbance is available when monoclonal antibodies the epitopes of whichare apart from each other are combined. The combination of anti-TSmonoclonal antibodies the epitopes of which are apart from each otherhas made it possible to detect TS by the sandwich method.

Capability of Exploitation in Industry

As has been described above, the anti-human TS monoclonal antibodiesaccording to the present invention are useful for the immunologicalmeasurement of human TS. Quantitation of human TS in a sample (forexample, a stomach tissue extract) by their use permits not onlydetermination of the presence or absence of a tumor, confirmation oftherapeutic effect, and the like but also provision of an indication asto which treatment method should be chosen and whether or notadministration of an antitumor drug is permissible.

What is claimed is:
 1. An anti-human thymidylate synthase monoclonal antibody capable of recognizing an epitope within a region from the 267^(th) to the 282^(nd) amino acid from an N-terminus in human thymidylate synthase.
 2. An anti-human thymidylate synthase monoclonal antibody capable of recognizing an epitope from the 267^(th) to the 282^(nd) amino acid from an N-terminus in human thymidylate synthase, which is produced by mouse hybridoma RTSMA1 (FERM BP-6404) or RTSMA2 (FERM BP-6402).
 3. A hybridoma capable of producing an anti-human thyvidylate synthase monoclonal antibody, wherein said antibody is capable of recognizing an epitope within a region from the 267^(th) to the 282^(nd) amino acid from an N-terminus in human thymidylate synthase.
 4. A hybridoma capable of producing an anti-human thymidylate synthase monoclonal antibody, wherein said hybridoma is mouse hybridoma RTSMA1 (FERM BP-6404) or RTSMA2 (FERM BP-6402).
 5. An anti-human thymidylate synthase monoclonal antibody capable of recognizing an epitope that is recognized by a monoclonal antibody that is produced by mouse hybridoma RTSMA1 (FERM BP-6404) or RTSMA2 (FERM BP-6402). 